One of the major goals of the Human Genome Project is to obtain a map of the entire genome that contains well-characterized DNA landmarks placed every 100 kilobase pairs (kb). For a haploid human genome of about three billion base pairs, this would require a minimum of 30,000 markers. The goal of Project 1 is to generate over 30,000 Sequence-Tagged Site (STS) markers, which will then be used to construct a 100 kb resolution Radiation Hybrid (RH) map of the genome (Project 2). Since it is desirable for these STSs to be as randomly distributed throughout the genome as possible, we plan to develop them from a number of different sources, including 9,000 from anonymous small-insert clones. Another 3,000 STSs will be developed from cloned inter-Alu PCR products from human genomic DNA. In order to integrate our maps with those of other investigators, we will also develop 3,000 STSs for published genetic markers. All these STSs will be developed in a similar way to those generated by Project 1 for the mapping of chromosome 4. This will involve the construction of clone libraries, preparation of DNAs from the clones, determination and analysis of the sequences of the cloned inserts, followed by the design and synthesis of oligonucleotides for each STS. All of the STSs will be tested with a series of control DNA tempates. When an STS can be uniquely assigned to a single human chromosome (using the somatic cell hybrids of the Corriel mapping panel #1), that STS will be transferred to the other Projects. In addition to obtaining the STSs from multiple sources, we will use several methods for scoring the presence or absence of the STSs during the generation of different kinds of whole genome maps by several Projects in the Center.